ARTICLE Received 11 Aug 2013 | Accepted 5 Feb 2014 | Published 3 Mar 2014
DOI: 10.1038/ncomms4388
Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-b signalling FangFang Zhou1,2, Yvette Drabsch2, Tim J.A. Dekker3, Amaya Garcia de Vinuesa2, Yihao Li2, Lukas J.A.C. Hawinkels2, Kelly-Ann Sheppard4, Marie-Jose´ Goumans2, Rodney B. Luwor5, Carlie J. de Vries6, Wilma E. Mesker3, Rob A.E.M. Tollenaar3, Peter Devilee7, Chris X. Lu4, Hongjian Zhu5, Long Zhang1 & Peter ten Dijke1,2
In advanced cancers, the TGF-b pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-b signalling. NR4A1 promotes TGF-b/SMAD signalling by facilitating AXIN2–RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-b-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-b-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-b-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-b signalling in breast cancer.
1 Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China. 2 Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands. 3 Department of Surgery, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands. 4 Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. 5 Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital 5th Floor Clinical Science Building, Parkville, Victoria 3050, Australia. 6 Academic Medical Center, K1-113, University of Amsterdam, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. 7 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. Correspondence and requests for materials should be addressed to L.Z. (email: [email protected]) or to P.T.D. (email: [email protected]).
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
T
ransforming growth factor (TGF)-b signalling plays critical roles in embryonic development and the maintenance of tissue homoeostasis in all metazoans1–3. TGF-b signals via specific complexes of type I and type II Ser/Thr kinase receptors. The activated TGF-b type I receptor (TbRI) induces SMAD2/3 phosphorylation; phosphorylated SMAD2/3 forms heterooligomers with SMAD4, which accumulate in the nucleus to regulate the expression of target genes4,5. SMAD7 functions as an inhibitory SMAD by recruiting the E3 ligase SMURF1/2 to the type I receptor and subsequently degrading TbRI and mitigating TGF-b signalling6,7. ARKADIA and RNF12 potentiate TGF-b signalling by targeting SMAD7 for polyubiquitination and degradation8,9. Deregulation of TGF-b activity can result in cancer development. In normal and premalignant cells, TGF-b enforces homoeostasis and suppresses tumour progression directly through cell autonomous regulation of apoptosis and growth arrest or indirectly through blockage of paracrine factor production in the tumour stroma. However, when cancer cells lose the TGF-b tumour suppressive responses, they can utilize TGF-b as a potent promoter of cell motility, invasion, metastasis and tumour stem cell maintenance1,10. NR4A1 (also named Nur77, TR3 or NGF-IB) is a member of the steroid/thyroid hormone receptor superfamily. It is a transcription factor and as an early response gene, it can be induced by many stimuli including serum, inflammatory factors, growth factors and stress in different cell types and organs11. NR4A1 was considered as a strong tumour suppressor because of its involvement in growth inhibition and induction of apoptosis12–15. Nevertheless, NR4A1 was also reported to be highly expressed in colon and pancreatic tumours. Knockdown of NR4A1 in these cancer cells resulted in inhibition of cell growth, induction of apoptosis and decreased angiogenesis16,17. These findings suggest that NR4A1 has both a tumour suppressive and pro-oncogenic effect in cancer development. In this study, we discovered NR4A1 expression as a highly potent TGF-b/SMAD signalling activator by inducing the degradation of SMAD7. Moreover, we demonstrate that inflammation-induced NR4A1 greatly potentiates TGF-b-induced breast cancer cell invasion and metastasis. Results NR4A1 is an activator of TGF-b/SMAD signalling. To identify novel regulators of TGF-b signalling, a genome-wide geneby-gene screen was conducted. The Origene10K (two sets) and MGC7K complementary DNA libraries18 (total 27,000 genes) were screened in duplicate in HEK293T cells stably transfected with the SMAD3/SMAD4-dependent CAGAR12Luc transcriptional reporter stimulated with a suboptimal dose of TGF-b. Among the screened cDNAs, nuclear receptor NR4A1 was identified as one of the top three activators of TGF-b/SMAD signalling (Fig. 1a and Supplementary Fig. 1). Consistently, in a short interfering RNA screen depleting 5,000 druggable genes one by one in the same reporter cell line, siNR4A1 was identified as an inhibitor of this response (Fig. 1b). NR4A1 promoted TGF-b/SMAD transcription to a greater extent compared with SMAD3, whereas short hairpin RNA (shRNA)-mediated NR4A1 depletion decreased TGF-b/SMAD transcription (Fig. 1c). Stably expressing NR4A1 in PC3 prostate cancer cells highly potentiated TGF-b-induced SMAD2 and SMAD3 phosphorylation, whereas PC3 cells with stable NR4A1 knockdown dramatically decreased these responses (Fig. 1d,e). Ectopic NR4A1 expression increased SMAD2/3–SMAD4 complex formation, even without TGF-b stimulation, whereas shRNA2
mediated NR4A1 depletion reduced complex formation (Supplementary Fig. 2a,b). SMAD2/3 nuclear translocation was enhanced by NR4A1 expression and diminished by NR4A1 depletion (Supplementary Fig. 2c). In addition, when ectopically expressed in HaCaT cells, NR4A1 promoted SMAD2 phosphorylation, even without the addition of TGF-b ligand. This result was due to autocrine TbRI activation because the TbRI kinase inhibitor compound (SB431542) blocked this induction (Fig. 1f). Moreover, subcutaneous injection of head and neck cancer, head and neck (HN)5 cells expressing NR4A1 led to the high SMAD3 transcriptional HN5 cell autonomous activity in vivo mediated by endogenous TGF-b-like ligands without significant difference in cell proliferation (Fig. 2a–c, Supplementary Fig. 3a,b and data not shown). Furthermore, phospho-SMAD2 was greatly induced in mouse livers infected with adenovirus expressing NR4A1, indicating in situ activation of the TGF-b signal by NR4A1 (Fig. 2d,e and Supplementary Fig. 3c). Compared with wild-type mouse embryonic fibroblasts (MEFs), TGF-b-induced phosphoSMAD2 was decreased in primary NR4A1 / MEFs (Fig. 2f). Consistent with this finding, two direct target genes of TGF-b/ SMAD, that is, SMAD7, which participates in a negative feedback loop and connective tissue growth factor (CTGF), were downregulated in NR4A1 / MEF cells compared with wild-type MEFs (Fig. 2g). In addition, in breast cancer patients, we observed strong and significant positive correlation between NR4A1 and TGF-b/SMAD direct target genes PAI-1 (plasminogen activator inhibitor-1)/CTGF, as well as weak but also significant correlation between NR4A1 and Smad7/CXCR4 (CXC chemokine receptor 4; Fig. 2h). Thus, NR4A1 greatly activates and is a strong and important, but not essential, determinant of TGF-b/SMAD signalling. NR4A1 accelerates SMAD7 degradation. Enhancement of phospho-SMAD2 by NR4A1 suggests NR4A1 acts upstream of SMAD2/3. In addition, we found that NR4A1 did not induce SMAD3, SMAD4 and ALK5 expression at the messenger RNA level. Thus, NR4A1 does not appear to cooperate with SMAD3 as a transcription factor, nor does it induce the expression of agonistic TGF-b receptor/SMAD signalling mediators, to facilitate TGF-b/SMAD signalling. We next tested whether NR4A1 misexpression affects TbRI levels at the plasma membrane, where signalling is initiated19. Upon ectopic expression with NR4A1, biotin-labelled cell surface TbRI displayed a prolonged half-life (Fig. 3a). In line with this, NR4A1 depletion in MDA-MB-231 cells led to lower cell surface TbRI levels and accelerated degradation (Fig. 3b). SMAD7 functions as an inhibitory SMAD by recruiting E3 ligase SMURF1/2 to the type I receptor and subsequently degrading TbRI and mitigating TGF-b signalling6,7. Indeed, in NR4A1 stably depleted PC3 cells, we found that the polyubiquitination of TbRI was increased (Supplementary Fig. 4a). Interestingly, NR4A1 did not bind to R-SMAD and SMAD4, but specifically bound inhibitory SMAD7 (Fig. 3c). This made us analyse the effect of NR4A1 on SMAD7 protein expression. SMAD7 protein (in contrast to SMAD7 mRNA) was downregulated in PC3 cells stably expressing NR4A1 (Fig. 3d). Conversely, in NR4A1 stably depleted cells, SMAD7 was upregulated. Consistent with this, we observed that TbRI inversely correlated with SMAD7 level, and was upregulated and downregulated in NR4A1 stably expressing and depleted cells, respectively (Fig. 3d). In HeLa cells, SMAD7 overexpression reduced biotin-labelled TbRI level, whereas NR4A1 co-expression inhibited SMAD7 expression and restored the membraneassociate TbRI level (Fig. 3e). Furthermore, we found that NR4A1-deficient MEFs showed higher expression levels and longer half-lives of SMAD7 than wild-type MEFs (Fig. 3f).
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
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Figure 1 | NR4A1 is screened as an activator of TGF-b/SMAD signalling. (a) A genome-wide cDNA overexpression screen in HEK 293T cells using TGF-b-induced CAGA12-luc transcriptional response as assay. Three NR4A1 cDNAs that potently stimulated the luciferase activity were identified. As expected, SMAD3 and SMAD7 stimulated and inhibited the luciferase activity, respectively. (b) Short interfering RNA (siRNA) screening chart for 5,000 druggable genes in which two NR4A1 siRNA were identified that repressed the luciferase activity. siALK5, siLuciferase and siSMAD3 that strongly repress luciferase activity served as positive controls. The x and y axes are the relative luciferase activity in two replicates in a and b. (c) Validation of NR4A1 overexpression or knockdown for SMAD3 transcriptional response induced by TGF-b (5 ng ml 1) in HEK293T cells. Data are presented as mean±s.d. (n ¼ 3 measurements). NS shRNA, non-targeting shRNA; RLU, relative luciferase units. (d) Immunoblot (IB) analysis of PC3 cells stably expressing NR4A1. (e) Immunoblot (IB) analysis of PC3 cells stably depleted of NR4A1 by shRNA (shNR4A1). (f) HaCaT cells stably expressing NR4A1 were treated with TGF-b (1 ng ml 1) or SB431542 (10 mM) for 1 h and then lysed for immunoblot analysis.
When we restored NR4A1 expression in NR4A1-deficient cells, SMAD7 expression was decreased and SMAD7 degradation was accelerated (Fig. 3g). As expected, in NR4A1-depleted cells, SMAD7 polyubiquitination was strongly diminished (Supplementary Fig. 4b). NR4A1 and SMAD7 co-localized in the nucleus, but only in the presence of MG132. Ectopic-expressed SMAD7 was degraded in HaCaT cells that stably expressed NR4A1; only in a few cells some cytoplasmic SMAD7 remained (Supplementary Fig. 4c). These results together suggest that NR4A1 potentiates TGF-b/SMAD signalling by facilitating SMAD7 degradation. SMAD7 is targeted for degradation by E3 ligases ARKADIA, RNF12 and also by AXIN1 (refs 8,9,20,21,22). We found that AXIN2 could also interact with endogenous ARKADIA, RNF12 and rely on them to target SMAD7 for polyubiquitination (Supplementary Fig. 4d,e). Immunoprecipitation studies showed that NR4A1 associated with the SMAD7/ARKADIA/RNF12/ AXIN2 complex endogenously but not with SMURFs (Fig. 3h). Importantly, as a scaffold protein for the SMAD7 destruction complex, endogenous AXIN2 (but not AXIN1) expression was sharply upregulated by NR4A1 (Fig. 3h). SMAD7 polyubiquitination was increased by ectopic NR4A1 expression. Co-expression of NR4A1 or AXIN2 with ARKADIA or RNF12 synergistically
enhanced the polyubiquitination of SMAD7 (Fig. 3i). Depletion of E3 ligase RNF12 or ARKADIA mitigated the SMAD7 polyubiquitination. Double depletion of RNF12 and ARKADIA decreased SMAD7 polyubiquitination to a basal level, indicating that NR4A1 enhances SMAD7 polyubiquitination mainly via both E3 ligases RNF12 and ARKADIA (Fig. 3j). Moreover, polyubiquitinated SMAD7 accumulated in NR4A1 þ / þ MEFs but was undetectable in NR4A1 / cells, although it was detected again on restoring the expression of NR4A1 or on overexpression of AXIN2 (Fig. 3k). Depleting AXIN2 greatly decreased NR4A1triggered endogenous SMAD7 polyubiquitination, suggesting that NR4A1 relies on the E3 ubiquitin ligase RNF12/ARKADIA and scaffold protein AXIN2 to degrade SMAD7. NR4A1 directly induces AXIN2. To explore the mechanism by which NR4A1 induces AXIN2, we first examined the endogenous AXIN2 protein expression in different mouse or human cell lines stably expressing NR4A1. AXIN2 was upregulated in mouse NMuMG and human HaCaT or PC3 cells (Fig. 4a). The AXIN2 protein level was lower in primary NR4A1 / MEFs than in control MEFs (Fig. 4b). The real-time quantitative (qRT)–PCR analysis showed that stable NR4A1 expression in HaCaT, PC3,
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
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DOI: 10.1038/ncomms4388
Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-b signalling FangFang Zhou1,2, Yvette Drabsch2, Tim J.A. Dekker3, Amaya Garcia de Vinuesa2, Yihao Li2, Lukas J.A.C. Hawinkels2, Kelly-Ann Sheppard4, Marie-Jose´ Goumans2, Rodney B. Luwor5, Carlie J. de Vries6, Wilma E. Mesker3, Rob A.E.M. Tollenaar3, Peter Devilee7, Chris X. Lu4, Hongjian Zhu5, Long Zhang1 & Peter ten Dijke1,2
In advanced cancers, the TGF-b pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-b signalling. NR4A1 promotes TGF-b/SMAD signalling by facilitating AXIN2–RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-b-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-b-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-b-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-b signalling in breast cancer.
1 Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China. 2 Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands. 3 Department of Surgery, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands. 4 Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. 5 Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital 5th Floor Clinical Science Building, Parkville, Victoria 3050, Australia. 6 Academic Medical Center, K1-113, University of Amsterdam, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. 7 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. Correspondence and requests for materials should be addressed to L.Z. (email: [email protected]) or to P.T.D. (email: [email protected]).
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
1
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
T
ransforming growth factor (TGF)-b signalling plays critical roles in embryonic development and the maintenance of tissue homoeostasis in all metazoans1–3. TGF-b signals via specific complexes of type I and type II Ser/Thr kinase receptors. The activated TGF-b type I receptor (TbRI) induces SMAD2/3 phosphorylation; phosphorylated SMAD2/3 forms heterooligomers with SMAD4, which accumulate in the nucleus to regulate the expression of target genes4,5. SMAD7 functions as an inhibitory SMAD by recruiting the E3 ligase SMURF1/2 to the type I receptor and subsequently degrading TbRI and mitigating TGF-b signalling6,7. ARKADIA and RNF12 potentiate TGF-b signalling by targeting SMAD7 for polyubiquitination and degradation8,9. Deregulation of TGF-b activity can result in cancer development. In normal and premalignant cells, TGF-b enforces homoeostasis and suppresses tumour progression directly through cell autonomous regulation of apoptosis and growth arrest or indirectly through blockage of paracrine factor production in the tumour stroma. However, when cancer cells lose the TGF-b tumour suppressive responses, they can utilize TGF-b as a potent promoter of cell motility, invasion, metastasis and tumour stem cell maintenance1,10. NR4A1 (also named Nur77, TR3 or NGF-IB) is a member of the steroid/thyroid hormone receptor superfamily. It is a transcription factor and as an early response gene, it can be induced by many stimuli including serum, inflammatory factors, growth factors and stress in different cell types and organs11. NR4A1 was considered as a strong tumour suppressor because of its involvement in growth inhibition and induction of apoptosis12–15. Nevertheless, NR4A1 was also reported to be highly expressed in colon and pancreatic tumours. Knockdown of NR4A1 in these cancer cells resulted in inhibition of cell growth, induction of apoptosis and decreased angiogenesis16,17. These findings suggest that NR4A1 has both a tumour suppressive and pro-oncogenic effect in cancer development. In this study, we discovered NR4A1 expression as a highly potent TGF-b/SMAD signalling activator by inducing the degradation of SMAD7. Moreover, we demonstrate that inflammation-induced NR4A1 greatly potentiates TGF-b-induced breast cancer cell invasion and metastasis. Results NR4A1 is an activator of TGF-b/SMAD signalling. To identify novel regulators of TGF-b signalling, a genome-wide geneby-gene screen was conducted. The Origene10K (two sets) and MGC7K complementary DNA libraries18 (total 27,000 genes) were screened in duplicate in HEK293T cells stably transfected with the SMAD3/SMAD4-dependent CAGAR12Luc transcriptional reporter stimulated with a suboptimal dose of TGF-b. Among the screened cDNAs, nuclear receptor NR4A1 was identified as one of the top three activators of TGF-b/SMAD signalling (Fig. 1a and Supplementary Fig. 1). Consistently, in a short interfering RNA screen depleting 5,000 druggable genes one by one in the same reporter cell line, siNR4A1 was identified as an inhibitor of this response (Fig. 1b). NR4A1 promoted TGF-b/SMAD transcription to a greater extent compared with SMAD3, whereas short hairpin RNA (shRNA)-mediated NR4A1 depletion decreased TGF-b/SMAD transcription (Fig. 1c). Stably expressing NR4A1 in PC3 prostate cancer cells highly potentiated TGF-b-induced SMAD2 and SMAD3 phosphorylation, whereas PC3 cells with stable NR4A1 knockdown dramatically decreased these responses (Fig. 1d,e). Ectopic NR4A1 expression increased SMAD2/3–SMAD4 complex formation, even without TGF-b stimulation, whereas shRNA2
mediated NR4A1 depletion reduced complex formation (Supplementary Fig. 2a,b). SMAD2/3 nuclear translocation was enhanced by NR4A1 expression and diminished by NR4A1 depletion (Supplementary Fig. 2c). In addition, when ectopically expressed in HaCaT cells, NR4A1 promoted SMAD2 phosphorylation, even without the addition of TGF-b ligand. This result was due to autocrine TbRI activation because the TbRI kinase inhibitor compound (SB431542) blocked this induction (Fig. 1f). Moreover, subcutaneous injection of head and neck cancer, head and neck (HN)5 cells expressing NR4A1 led to the high SMAD3 transcriptional HN5 cell autonomous activity in vivo mediated by endogenous TGF-b-like ligands without significant difference in cell proliferation (Fig. 2a–c, Supplementary Fig. 3a,b and data not shown). Furthermore, phospho-SMAD2 was greatly induced in mouse livers infected with adenovirus expressing NR4A1, indicating in situ activation of the TGF-b signal by NR4A1 (Fig. 2d,e and Supplementary Fig. 3c). Compared with wild-type mouse embryonic fibroblasts (MEFs), TGF-b-induced phosphoSMAD2 was decreased in primary NR4A1 / MEFs (Fig. 2f). Consistent with this finding, two direct target genes of TGF-b/ SMAD, that is, SMAD7, which participates in a negative feedback loop and connective tissue growth factor (CTGF), were downregulated in NR4A1 / MEF cells compared with wild-type MEFs (Fig. 2g). In addition, in breast cancer patients, we observed strong and significant positive correlation between NR4A1 and TGF-b/SMAD direct target genes PAI-1 (plasminogen activator inhibitor-1)/CTGF, as well as weak but also significant correlation between NR4A1 and Smad7/CXCR4 (CXC chemokine receptor 4; Fig. 2h). Thus, NR4A1 greatly activates and is a strong and important, but not essential, determinant of TGF-b/SMAD signalling. NR4A1 accelerates SMAD7 degradation. Enhancement of phospho-SMAD2 by NR4A1 suggests NR4A1 acts upstream of SMAD2/3. In addition, we found that NR4A1 did not induce SMAD3, SMAD4 and ALK5 expression at the messenger RNA level. Thus, NR4A1 does not appear to cooperate with SMAD3 as a transcription factor, nor does it induce the expression of agonistic TGF-b receptor/SMAD signalling mediators, to facilitate TGF-b/SMAD signalling. We next tested whether NR4A1 misexpression affects TbRI levels at the plasma membrane, where signalling is initiated19. Upon ectopic expression with NR4A1, biotin-labelled cell surface TbRI displayed a prolonged half-life (Fig. 3a). In line with this, NR4A1 depletion in MDA-MB-231 cells led to lower cell surface TbRI levels and accelerated degradation (Fig. 3b). SMAD7 functions as an inhibitory SMAD by recruiting E3 ligase SMURF1/2 to the type I receptor and subsequently degrading TbRI and mitigating TGF-b signalling6,7. Indeed, in NR4A1 stably depleted PC3 cells, we found that the polyubiquitination of TbRI was increased (Supplementary Fig. 4a). Interestingly, NR4A1 did not bind to R-SMAD and SMAD4, but specifically bound inhibitory SMAD7 (Fig. 3c). This made us analyse the effect of NR4A1 on SMAD7 protein expression. SMAD7 protein (in contrast to SMAD7 mRNA) was downregulated in PC3 cells stably expressing NR4A1 (Fig. 3d). Conversely, in NR4A1 stably depleted cells, SMAD7 was upregulated. Consistent with this, we observed that TbRI inversely correlated with SMAD7 level, and was upregulated and downregulated in NR4A1 stably expressing and depleted cells, respectively (Fig. 3d). In HeLa cells, SMAD7 overexpression reduced biotin-labelled TbRI level, whereas NR4A1 co-expression inhibited SMAD7 expression and restored the membraneassociate TbRI level (Fig. 3e). Furthermore, we found that NR4A1-deficient MEFs showed higher expression levels and longer half-lives of SMAD7 than wild-type MEFs (Fig. 3f).
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
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Figure 1 | NR4A1 is screened as an activator of TGF-b/SMAD signalling. (a) A genome-wide cDNA overexpression screen in HEK 293T cells using TGF-b-induced CAGA12-luc transcriptional response as assay. Three NR4A1 cDNAs that potently stimulated the luciferase activity were identified. As expected, SMAD3 and SMAD7 stimulated and inhibited the luciferase activity, respectively. (b) Short interfering RNA (siRNA) screening chart for 5,000 druggable genes in which two NR4A1 siRNA were identified that repressed the luciferase activity. siALK5, siLuciferase and siSMAD3 that strongly repress luciferase activity served as positive controls. The x and y axes are the relative luciferase activity in two replicates in a and b. (c) Validation of NR4A1 overexpression or knockdown for SMAD3 transcriptional response induced by TGF-b (5 ng ml 1) in HEK293T cells. Data are presented as mean±s.d. (n ¼ 3 measurements). NS shRNA, non-targeting shRNA; RLU, relative luciferase units. (d) Immunoblot (IB) analysis of PC3 cells stably expressing NR4A1. (e) Immunoblot (IB) analysis of PC3 cells stably depleted of NR4A1 by shRNA (shNR4A1). (f) HaCaT cells stably expressing NR4A1 were treated with TGF-b (1 ng ml 1) or SB431542 (10 mM) for 1 h and then lysed for immunoblot analysis.
When we restored NR4A1 expression in NR4A1-deficient cells, SMAD7 expression was decreased and SMAD7 degradation was accelerated (Fig. 3g). As expected, in NR4A1-depleted cells, SMAD7 polyubiquitination was strongly diminished (Supplementary Fig. 4b). NR4A1 and SMAD7 co-localized in the nucleus, but only in the presence of MG132. Ectopic-expressed SMAD7 was degraded in HaCaT cells that stably expressed NR4A1; only in a few cells some cytoplasmic SMAD7 remained (Supplementary Fig. 4c). These results together suggest that NR4A1 potentiates TGF-b/SMAD signalling by facilitating SMAD7 degradation. SMAD7 is targeted for degradation by E3 ligases ARKADIA, RNF12 and also by AXIN1 (refs 8,9,20,21,22). We found that AXIN2 could also interact with endogenous ARKADIA, RNF12 and rely on them to target SMAD7 for polyubiquitination (Supplementary Fig. 4d,e). Immunoprecipitation studies showed that NR4A1 associated with the SMAD7/ARKADIA/RNF12/ AXIN2 complex endogenously but not with SMURFs (Fig. 3h). Importantly, as a scaffold protein for the SMAD7 destruction complex, endogenous AXIN2 (but not AXIN1) expression was sharply upregulated by NR4A1 (Fig. 3h). SMAD7 polyubiquitination was increased by ectopic NR4A1 expression. Co-expression of NR4A1 or AXIN2 with ARKADIA or RNF12 synergistically
enhanced the polyubiquitination of SMAD7 (Fig. 3i). Depletion of E3 ligase RNF12 or ARKADIA mitigated the SMAD7 polyubiquitination. Double depletion of RNF12 and ARKADIA decreased SMAD7 polyubiquitination to a basal level, indicating that NR4A1 enhances SMAD7 polyubiquitination mainly via both E3 ligases RNF12 and ARKADIA (Fig. 3j). Moreover, polyubiquitinated SMAD7 accumulated in NR4A1 þ / þ MEFs but was undetectable in NR4A1 / cells, although it was detected again on restoring the expression of NR4A1 or on overexpression of AXIN2 (Fig. 3k). Depleting AXIN2 greatly decreased NR4A1triggered endogenous SMAD7 polyubiquitination, suggesting that NR4A1 relies on the E3 ubiquitin ligase RNF12/ARKADIA and scaffold protein AXIN2 to degrade SMAD7. NR4A1 directly induces AXIN2. To explore the mechanism by which NR4A1 induces AXIN2, we first examined the endogenous AXIN2 protein expression in different mouse or human cell lines stably expressing NR4A1. AXIN2 was upregulated in mouse NMuMG and human HaCaT or PC3 cells (Fig. 4a). The AXIN2 protein level was lower in primary NR4A1 / MEFs than in control MEFs (Fig. 4b). The real-time quantitative (qRT)–PCR analysis showed that stable NR4A1 expression in HaCaT, PC3,
NATURE COMMUNICATIONS | 5:3388 | DOI: 10.1038/ncomms4388 | www.nature.com/naturecommunications
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4388
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